We will continue our studies of the human apolipoprotein genes to determine what role genetic variation in them might play in atherosclerosis susceptibility. Our major focus will be on the apo A-I and apo E genes. There are two specific aims: 1) Transcriptional regulation of apolipoprotein gene expression: The cis-Acting DNA elements that control transcription of the apo A-I, E and CIII genes will be defined. Each of the cloned genes will be manipulated in vitro and then transfected into differentiated human cell cultures. Transient expression will be assessed by either RNA or protein analysis. Transcriptionally active DNA elements will be defined by deletion analysis and by site specific mutagenesis. Initially, apolipoprotein gene expressing HepG2 and non-expressing Hela cells will be used, followed by studies in macrophage and intestinal cell lines. Trans-acting protein factors that bind to these regions will be characterized and purified using mobility shift and in vitro transcription assays. Finally, the transgenic mouse model will be used to verify in vivo the DNA sequence requirements for tissue specific expression. In this model we will also test whether apo A-I gene transcriptional regulation affects plasma HDL levels. 2) Clinically significant apolipoprotein genetic variation. RFLP's at the chromosome 11 (apo A-I) and chromosome 19 (apo E) gene loci will be assessed for association with myocardial infarction or its associated lipid, lipoprotein or apolipoprotein abnormalities. In addition, these RFLP's will be used in studying families with dyslipoproteinemia phenotypes to see if either of these loci are linked to particular clinical disorders. Partial or complete genomic libraries in plasmid or lambda vectors will be constructed from individuals who may have as their underlying defect a mutation in the apo A-I or apo E genes to isolate the gene harboring the suspected mutation. Structural and functional studies will be conducted to determine the exact nature of the mutation. It will then be possible to probe for the specific gene defect in the general population or in a subset with a particular clinical phenotype to determine the frequency of the mutation. This information could form the basis of new tests for atherosclerosis susceptibility in presymptomatic individuals.